Cut‐off values and significance of Oil Red O‐positive cells in bronchoalveolar lavage fluid

C. Basset‐Léobon, L. Lacoste‐Collin, J. Aziza, J.C. Bes, S. Jozan and M. Courtade‐Saïdi
Cut‐off values and significance of Oil Red O‐positive cells in bronchoalveolar lavage fluid Objective: To evaluate the percentage and predictive value of Oil Red O‐positive macrophages (ORO‐PM) to identify lipid‐laden macrophages in bronchoalveolar lavage fluids (BALF) from patients with different pathologies. Methods: The percentage and absolute numbers of ORO‐PM were evaluated in 305 BALF. The patients were separated into ten groups: corticosteroid treatment (n = 18), amiodarone treatment (n = 8), interstitial fibrosis (n = 11), human immunodeficiency virus (HIV)‐positive (n = 25), infectious pneumonia (n = 43), severe haematological disorder (n = 25), interstitial syndrome (n = 109), suspicion of cancer (n = 17), transplant recipients (n = 50) and controls (n = 43). The total and differential cell counts in BALF were recorded. The presence of specific pathogens was also noted. Parametric and non‐parametric tests were used to compare the values between groups. Receiver–operating characteristics (ROC) curves were established in order to determine a cut‐off value. Results: The percentages of ORO‐PM were (mean ± standard deviation) 21.67 ± 29.12 in the corticosteroid group, 10.00 ± 12.49 in the amiodarone group, 19.45 ± 20.72 in the interstitial fibrosis group, 47.80 ± 30.46 in the HIV group, 19.72 ± 26.26 in the infectious pneumonia group, 27.42 ± 30.04 in the severe haematological disorder group, 25.18 ± 30.63 in the interstitial syndrome group, 17.64 ± 27.76 in the suspicion of cancer group, 22.50 ± 27.27 in the transplanted recipients group and 2.63 ± 3.48 in the control group. Significantly higher values were found in all groups when compared with the control group (P < 0.001). Only the HIV group showed higher numbers of ORO‐PM when compared with the interstitial syndrome group (P < 0.01). According to ROC curves, > 6% ORO‐PM was suggested as the positive cut‐off value. Conclusion: Significantly increased numbers of ORO‐PM were associated with various lung pathologies. However, the higher numbers observed in HIV patients require further investigations.     

Prevalence,molecular characteristics and risk factors for cryptosporidiosis among Iranian immunocompromised patients

Cryptosporidium spp. is a major cause of diarrhea in developing countries, mainly affecting people with compromised immune systems in general and HIV‐infected individuals with low CD4 + T‐cell counts in particular. This infection is self‐limiting in healthy persons; however, it can be severe, progressive and persistent in those who are immunocompromised. There are few published studies concerning cryptosporidiosis and Cryptosporidium genotypes in Iranian immunocompromised patients and none of them describe risk factors. This study was undertaken to identify prevalence, genotypes and risk factors for cryptosporidiosis in immunocompromised patients. Three fecal samples were obtained at two day intervals from each of the 183 patients and processed with modified Ziehl–Neelsen staining methods and 18S rRNA gene amplification and sequencing. The overall infection prevalence was 6%. Cryptosporidium parvum was identified in isolates from five HIV‐infected patients, one patient who had undergone bone marrow transplantation and one with chronic lymphocytic leukemia. Cryptosporidium hominis was identified in isolates from two HIV‐infected patients and two patients with acute lymphocytic leukemia. According to univariate analysis, the statistically significant factors were diarrhea (OR = 21.7, CI = 2.83–78.4, P= 0.003), CD4 + lymphocytes less than 100 cells/mm³ (OR = 41.3, CI = 13.45–114.8, P < 0.0001), other microbial infections (OR = 7.1321.7, CI = 1.97–25.73, P = 0.006), weight loss (OR = 73.78, CI = 15.5–350, P < 0.0001), abdominal pain (OR = 10.29, CI = 2.81–37.74.4, P= 0.001), dehydration (OR = 72.1, CI = 17.6–341.5, P < 0.0001), vomiting (OR = 4.87, CI = 1.4–16.9, P= 0.015), nausea (OR = 9.4, CI = 2.38–37.2, P < 0.001), highly active antiretroviral therapy (OR = 0.089, CI = 0.01–0.8, P= 0.015) and diarrhea in household members (OR = 7.37, CI = 2.04–26.66, P= 0.001). After multivariate analysis and a backward deletion process, only < 100 CD4 + T‐lymphocytes/mm³ maintained a significant association with infection. The authors recommend that this infection should be suspected in patients with diarrhea, weight loss and dehydration in general and in diarrheal individuals with < 100 CD4 + T‐lymphocytes/mm³.     

Mitochondrial toxicity and caspase activation in HIV pregnant women

To assess the impact of HIV‐infection and highly active anti‐retroviral treatment in mitochondria and apoptotic activation of caspases during pregnancy and their association with adverse perinatal outcome. Changes of mitochondrial parameters and apoptotic caspase activation in maternal peripheral blood mononuclear cells were compared at first trimester of pregnancy and delivery in 27 HIV‐infected and ‐treated pregnant women versus 24 uninfected pregnant controls. We correlated immunovirological, therapeutic and perinatal outcome with experimental findings: mitochondrial DNA (mtDNA) content, mitochondrial protein synthesis, mitochondrial function and apoptotic caspase activation. The HIV pregnancies showed increased adverse perinatal outcome (OR: 4.81 [1.14–20.16]; P < 0.05) and decreased mtDNA content (42.66 ± 5.94%, P < 0.01) compared to controls, even higher in naïve participants. This depletion caused a correlated decrease in mitochondrial protein synthesis (12.82 ± 5.73%, P < 0.01) and function (20.50 ± 10.14%, P < 0.001), not observed in controls. Along pregnancy, apoptotic caspase‐3 activation increased 63.64 ± 45.45% in controls (P < 0.001) and 100.00 ± 47.37% in HIV‐pregnancies (P < 0.001), in correlation with longer exposure to nucleoside analogues. HIV‐infected women showed increased obstetric problems and declined genetic and functional mitochondrial parameters during pregnancy, especially those firstly exposed to anti‐retrovirals. The apoptotic activation of caspases along pregnancy is emphasized in HIV pregnancies promoted by nucleoside analogues. However, we could not demonstrate direct mitochondrial or apoptotic implication in adverse obstetric outcome probably because of the reduced sample size.     

Interaction of synthetic peptide octarphin (TPLVTLFK) with human blood lymphocytes

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of non‐opioid β‐endorphin receptor, was labeled with tritium to specific activity of 29 Ci/mmol. The analysis of [³H]octarphin binding to human T and B lymphocytes separated from normal human blood revealed the existence of one type of high‐affinity binding sites (receptors): K_d 3.0 and 3.2 nM, respectively. Besides unlabeled octarphin, unlabeled β‐endorphin possessed the ability to inhibit the specific binding of [³H]octarphin to Т and B lymphocytes (K_i 1.9 and 2.2 nМ, respectively). Tests of the specificity of the receptors revealed that they are not sensitive to naloxone, α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin. Thus, both T and B lymphocytes from normal human blood express non‐opioid receptor for β‐endorphin. Binding of the hormone to the receptor provides a fragment 12–19. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Stereoselective pharmacokinetics of stable isotope (+/−)‐[13C]‐pantoprazole: Implications for a rapid screening phenotype test of CYP2C19 activity

Aims: We have previously shown that the (±)‐[¹³C]‐pantoprazole breath test is a promising noninvasive probe of CYP2C19 activity. As part of that trial, plasma, breath test indices and CYP2C19 (*2, *3, and *17) genotype were collected. Here, we examined whether [¹³C]‐pantoprazole exhibits enantioselective pharmacokinetics and whether this enantioselectivity is correlated with indices of breath test. Methods: Plasma (−)‐ and (+)‐[¹³C]‐pantoprazole that were measured using a chiral HPLC were compared between CYP2C19 genotypes and correlated with breath test indices. Results: The AUC_(0‐∞) of (+)‐[¹³C]‐pantoprazole in PM (*2/*2, n = 4) was 10.1‐ and 5.6‐fold higher that EM (*1/*1or *17, n = 10) and IM (*1/*2or *3, n = 10) of CYP2C19, respectively (P < 0.001). The AUC_(0‐∞) of (−)‐[¹³C]‐pantoprazole only significantly differed between PMs and EMs (1.98‐fold; P = 0.05). The AUC_(0‐∞) ratio of (+)‐/(−)‐[¹³C]‐pantoprazole was 3.45, 0.77, and 0.67 in PM, IM, and EM genotypes, respectively. Breath test index, delta over baseline show significant correlation with AUC_(0‐∞) of (+)‐[¹³C]‐pantoprazole (Pearson's r = 0.62; P < 0.001). Conclusions: [¹³C]‐pantoprazole exhibits enantioselective elimination. (+)‐[¹³C]‐pantoprazole is more dependent on CYP2C19 metabolic status and may serve as a more attractive probe of CYP2C19 activity than (−)‐[¹³C]‐pantoprazole or the racemic mixture. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Correlation Between Pneumocystis jirovecii Mitochondrial Genotypes and High and Low Fungal Loads Assessed by Single Nucleotide Primer Extension Assay and Quantitative Real‐Time PCR

We designed a single nucleotide primer extension (SNaPshot) assay for Pneumocystis jirovecii genotyping, targeting mt85 SNP of the mitochondrial large subunit ribosomal RNA locus, to improve minority allele detection. We then analyzed 133 consecutive bronchoalveolar lavage (BAL) fluids tested positive for P. jirovecii DNA by quantitative real‐time PCR, obtained from two hospitals in different locations (Hospital 1 [n = 95] and Hospital 2 [n = 38]). We detected three different alleles, either singly (mt85C: 39.1%; mt85T: 24.1%; mt85A: 9.8%) or together (27%), and an association between P. jirovecii mt85 genotype and the patient's place of hospitalization (p = 0.011). The lowest fungal loads (median = 0.82 × 10³ copies/μl; range: 15–11 × 10³) were associated with mt85A and the highest (median = 1.4 × 10⁶ copies/μl; range: 17 × 10³–1.3 × 10⁷) with mt85CTA (p = 0.010). The ratios of the various alleles differed between the 36 mixed‐genotype samples. In tests of serial BALs (median: 20 d; range 4–525) from six patients with mixed genotypes, allele ratio changes were observed five times and genotype replacement once. Therefore, allele ratio changes seem more frequent than genotype replacement when using a SNaPshot assay more sensitive for detecting minority alleles than Sanger sequencing. Moreover, because microscopy detects only high fungal loads, the selection of microscopy‐positive samples may miss genotypes associated with low loads.     

Respirable powder formulation of a shortened vasoactive intestinal peptide analog for treatment of airway inflammatory diseases

The aim of present study was to develop a respirable powder (RP) of a shortened vasoactive intestinal peptide (VIP) analog for inhalation. VIP and C‐terminally truncated VIP analogs were synthesized with a solid‐phase method. A structure‐activity relationship (SAR) study was carried out in terms with binding and relaxant activities of the peptides. Prepared RP formulation of a shortened VIP analog was physicochemically characterized by morphological, in vitro aerodynamic, and pharmacological assessments. The SAR study demonstrated that the N‐terminal 23 amino acid residues were required for biological activity of VIP. Upon chemical modification of VIP(1–23), [R^{15, 20, 21}, L¹⁷]‐VIP(1–23) was newly developed, which had higher binding activity in rat lung and smooth muscle relaxant effect in mouse stomach than VIP(1–23). The [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)‐based RP, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP, exhibited fine in vitro inhalation performance. Airway inflammation evoked by sensitization of antigen in rats was attenuated by pre‐treatment with the [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP at a dose of 50 μg‐[R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/rat as evidenced by a 70% reduction of recruited inflammatory cells in bronchoalveolar lavage fluid. On the basis of these results, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP might be a promising agent for treatment of airway inflammatory diseases.     

Poster Sessions BP01: Energy Metabolism. NMR investigation of metabolic alterations in the brain of thiamine‐deficient rats

Thiamine deficiency (TD) results in region‐selective impairment of brain metabolism. Since thiamine is a cofactor for enzymes involved in glucose metabolism, ¹H and ¹³C‐NMR was used to investigate metabolic fluxes through the major pathways of glucose metabolism in vulnerable (medial thalamus, MT; inferior colliculus, IC) and nonvulnerable brain structures of rats made thiamine deficient following treatment with the central thiamine antagonist pyrithiamine vs. pair‐fed controls. Symptomatic stages of TD resulted in decreased glutamate and GABA in MT an IC confirming previous biochemical studies. ¹³C‐isotopomer analysis revealed decreased de novo synthesis of [4–¹³C]glutamate (30%p < 0.02) and [2–¹³C]GABA (60%p < 0.01) in MT and IC consistent with decreased activities of pyruvate‐ and α‐ketoglutarate dehydrogenases. These changes were accompanied by decreased consumption of glucose and increased synthesis of lactate from [1–¹³C]glucose confirming decreased mitochondrial metabolism. Accumulation of glyceraldehyde‐3‐phosphate suggested inhibition of glucose flux through the thiamine‐deficient enzyme transketolase. Onset of symptoms of TD and significant cell death was accompanied by decreased neuronal marker molecules NAA and NAAG in MT. Focal lactate accumulation resulting from decreased activities of mitochondrial thiamine‐dependent enzymes appears to play a key role in the pathogenesis of selective neuronal cell death in TD. [funded by CIHR Canada].     

Maintenance costs of serotiny do not explain weak serotiny

Considerable variation in the duration of serotiny exists among species of both Australian and South African Proteaceae. ‘Weak’ serotiny (pre‐fire loss after <3 years) could be dictated by the costs (water or carbon) of cone/fruit retention or by benefits accruing from pre‐fire seed establishment. We determined that cones/fruits of a range of Australian and south western Cape Proteaceae species (Leucadendron xanthoconus, Aulax umbellata, L. linifolium, L. gandogeri, Hakea drupacea, H. sericea) are not sealed dead wood, but that they continuously lose H₂O and CO₂. Water loss from cones/fruits was poorly controlled, occurring in both light and dark. The rates of both H₂O and CO₂ loss from mature cones/fruits were negatively correlated with the degree of serotiny (r² = 0.59 and 0.18, respectively, P < 0.001 both). However, the amounts of H₂O and CO₂ lost per weight were small relative to the fluxes from leaves (13–29% for H₂O and 3–10% for CO₂). The [N] and [P] in the cones/fruits and seeds was substantial. Despite 25% of N and 38% of P being recovered from the cones/fruits following maturation, the loss of the cones/fruits and seeds would still incur a substantial nutrient cost. The seed [P] was positively correlated with the degree of serotiny (r² = 0.24, P = 0.001). We suggest that maintenance costs (water and carbon) of serotiny, although exceeding those of soil stored seeds, are relatively low. The correlation between the degree of serotiny and seed [P] indicates that stronger serotiny is required, much like sclerophylly, for survival under low nutrient availability in frequently burnt vegetation.     

Association of interleukin‐27 gene polymorphisms with susceptibility to HIV infection and disease progression

Interleukin‐27 (IL‐27) gene polymorphisms are linked to infectious disease susceptibility and IL‐27 plasma level is associated with HIV infection. Therefore, we aimed to investigate the association between IL‐27 polymorphisms and susceptibility to HIV infection and disease progression. A total of 300 patients with HIV infection (48 long‐term nonprogressors and 252 typical progressors) and 300 healthy controls were genotyped for three IL‐27 polymorphisms, rs17855750, rs181206, rs40837 which were performed by using multiple single nucleotide primer extension technique. Significant association was found between IL‐27 rs40837 polymorphisms with susceptibility to HIV infection (AG vs AA: adjusted OR = 1.60, 95% CI, 1.11‐2.30, P = 0.012; AG+GG vs AA: adjusted OR = 1.44, 95% CI, 1.02‐2.03, P = 0.038) and disease progression (LTNP: AG vs AA: adjusted OR = 2.33, 95% CI, 1.13‐4.80, P = 0.021; TP: AG vs AA: adjusted OR = 1.50, 95% CI, 1.04‐2.24, P = 0.030). Serum IL‐27 levels were significantly lower in cases compared to controls (P < 0.001). There were lower serum IL‐27 levels in TPs than in LTNPs (P < 0.001). We further found that LTNPs with rs40837 AG or GG genotype had lower serum IL‐27 levels than with AA genotype (P < 0.05). The CD4⁺T counts in cases were significantly lower than controls (P < 0.001). In contrast, individuals with rs40837 AG genotype had lower CD4⁺T counts than with AA genotype in cases (P < 0.05). In addition, CD4⁺T counts in TPs were significantly lower than LTNPs (P < 0.001). IL‐27 rs40837 polymorphism might influence the susceptibility to HIV infection and disease progression probably by regulating the level of serum IL‐27 or the quantity of CD4⁺T.     

Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes

Plasma membranes (1–2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 μM [α-³²P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37°C for 1 h; during this incubation, the [³²P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [³²P] guanosine diphosphate (GDP). [³²P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ maximally inhibited GDP release by 80–90%, whereas, 5 mM Cu²⁺ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One μM Gpp(NH)p (5′-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [³²P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1–100 nM glucagon (used as a positive control) stimulated [³²P]GDP release by about 17% (P < .05); similarly, 0.1–100 nM insulin stimulated [³²P]GDP release by 10–13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [³²P]GDP release by 7–10%. In the rat membranes, 10 nM insulin stimulated [³²P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [³²P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 μg of insulin receptor antibody stimulated [³²P]GDP release by 12–22% (P < .05) and 7–14% in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide-binding transducer protein (G protein) and activation of GTP/GDP exchange.     

Time-of-day effects of exposure to solar radiation on thermoregulation during outdoor exercise in the heat

High solar radiation has been recognised as a contributing factor to exertional heat-related illness in individuals exercising outdoors in the heat. Although solar radiation intensity has been known to have similar time-of-day variation as body temperature, the relationship between fluctuations in solar radiation associated with diurnal change in the angle of sunlight and thermoregulatory responses in individuals exercising outdoors in a hot environment remains largely unknown. The present study therefore investigated the time-of-day effects of variations in solar radiation associated with changing solar elevation angle on thermoregulatory responses during moderate-intensity outdoor exercise in the heat of summer. Eight healthy, high school baseball players, heat-acclimatised male volunteers completed a 3-h outdoor baseball trainings under the clear sky in the heat. The trainings were commenced at 0900 h in AM trial and at 1600 h in PM trial each on a separate day. Solar radiation and solar elevation angle during exercise continued to increase in AM (672–1107 W/m² and 44–69°) and decrease in PM (717–0 W/m² and 34–0°) and were higher on AM than on PM (both P < 0.001). Although ambient temperature (AM 32–36°C, PM 36–30°C) and wet-bulb globe temperature (AM 31–33°C, PM 34–27°C) also continued to increase in AM and decrease in PM, there were no differences between trials in these (both P > 0.05). Tympanic temperature measured by an infrared tympanic thermometer and mean skin temperature were higher in AM than PM at 120 and 180 min (P < 0.05). Skin temperature was higher in AM than PM at the upper arm and thigh at 120 min (P < 0.05) and at the calf at 120 and 180 min (both P < 0.05). Body heat gain from the sun was greater during exercise in AM than PM (P < 0.0001), at 0–60 min in PM than AM (P < 0.0001) and at 120–180 min in AM than PM (P < 0.0001). Dry heat loss during exercise was greater at 0–60 min (P < 0.0001), and lower at 60–120 min (P < 0.05) and 120–180 min (P < 0.0001) in AM than PM. Evaporative heat loss during exercise was greater in PM than AM at 120–180 min (P < 0.0001). Total (dry + evaporation) heat loss at the skin was greater during exercise in PM than AM (P < 0.0001), at 0–60 min in AM than PM (P < 0.0001) and at 60–120 and 120–180 min in PM than AM (P < 0.05 and 0.0001). Heart rate at 120–150 min was also higher in AM than PM (P < 0.05). Neither perceived thermal sensation nor rating of perceived exertion was different between trials (both P > 0.05). The current study demonstrates a greater thermoregulatory strain in the morning than in the afternoon resulting from a higher body temperature and heart rate in relation to an increase in environmental heat stress with rising solar radiation and solar elevation angle during moderate-intensity outdoor exercise in the heat. This response is associated with a lesser net heat loss at the skin and a greater body heat gain from the sun in the morning compared with the afternoon.     

Evidence of <Emphasis Type="Italic">Pneumocystis jiroveci</Emphasis> in human clinical samples in southwestern Slovakia over a 10-year period (2001–2010)

In the past, Pneumocystis jiroveci (formerly P. carinii) belonged to the Protozoa group, but the studies on structure of the cell wall and nucleotide sequence resulted in the reclassification of this organism in the kingdom Fungi. P. jiroveci is an opportunistic pathogen, responsible for pneumocystis pneumonia with frequent complications of immunocompromised patients. Delayed initiation of appropriate therapy increases the risk of death in immunocompromised patient. The aim of this work was to determine the prevalence of P. jiroveci from patients suspected of having respiratory tract infections in southwestern Slovakia over a 10-year period. Due to the increasing number of immunosuppressed persons, the diagnostic of P. jiroveci in patients with pulmonary complications is essential to improve recovery onsets. Effective diagnosis is currently based on microscopic examination and detection of parasite DNA by polymerase chain reaction (PCR) in bronchoalveolar lavage (BAL) and induced sputum. In total, 386 clinical samples originated from patients suspected of pneumocystis infection were tested within ten years. Requirements for diagnosis of the pathogen were growing during the period. Three hundred and sixteen BALs, 59 induced sputa, 10 lung biopsies and 1 liquor were subjected to the detection of P. jiroveci. P. jiroveci DNA was detected in 30 patients using PCR, but cysts of microorganism were present only in 4 cases by microscopy. The pathogen was confirmed in 24 BALs and 6 sputa samples. The presence of P. jiroveci has been demonstrated mainly in immunocompromised individuals with cancer (20), but also in patients with pneumonia (6+1 HIV), with unspecified parasitic diseases (1+1HIV) and with systemic lupus erythematosus (1).     

Leaf area estimation in a sugar beet cultivar by linear models

An indirect method of leaf area measurement for Rizor sugar beet cultivar was tested. Leaves were sampled during two growing seasons in a Randomised Complete Block Design experiment. For 2002 samplings, leaf area [cm²] was linearly correlated with maximum leaf width [cm] using all leaf samples (r ² = 0.83, p < 0.001) or using the means of the 8 sampling occasions (r ² = 0.97, p < 0.001). Correlations between leaf area and leaf mid vein length [cm] were weaker (r ² = 0.75, p < 0.001 and r ² = 0.93, p < 0. 001, respectively). For 2003 samplings, the area estimated by the equations was highly correlated to the measured leaf area.     

Pluronic F‐68 Enhanced Shoot Regeneration in Micropropagated Citrus Rootstock and Passiflora Species

The promotory effects have been studied of the non‐ionic surfactant, Pluronic F‐68, on bud induction/shoot regeneration in epicotyl and cotyledon explants of Citrus depressa and on shoot regeneration from leaf segments of 4–6 week‐old axenic nodal segment‐derived in vitro plants of Passiflora mollissima, P. giberti and P. edulis var. flavicarpa. For epicotyls of C. depressa, supplementation of agar‐solidified MS‐based bud induction/shoot regeneration medium with 0.5% [w/v] Pluronic F‐68 significantly (P < 0.05) increased mean fresh weight gain of cultures, percentage of explants giving shoots and number of shoots per explant. The same Pluronic concentration also enhanced the mean percentage of cotyledons exhibiting bud induction and the number of buds regenerated per cotyledon explant. Fresh weight gain was unaffected across the range of concentrations (0.001–0.5% w/v) of Pluronic F‐68 evaluated for this latter explant source. For leaf explants from axenic shoot cultures of P. mollissima, supplementation of NN‐based medium, containing 3 mg/l 6‐benzyladenine and 2.0 mg/l kinetin with 0.001–0.5% [w/v] Pluronic F‐68, significantly (P < 0.05) increased mean (± s.e.m.) biomass gain by a maximum of 2.7 ± 0.1 g fresh weight (g f.wt.) over the control. Similarly, for leaf explants of P. giberti, 0.001–0.5% [w/v] Pluronic F‐68 in MS‐based medium, containing 1.0 mg/l 6‐BAP and 0.5 mg/l kinetin significantly (P < 0.05) increased mean percentage of explants undergoing shoot regeneration. For P. edulis leaf explants, mean f.wt. gain was also significantly (P < 0.05) higher with Pluronic F‐68 at 0.001–0.5% [w/v].     

Selective effects of hydrocortisone on intestinal lipoprotein and apolipoprotein synthesis in the human fetus

Studies employing human fetal intestine have yielded much interesting information on the role of polarized enterocytes in fat absorption and transport. Using the organ culture model, we examined the influence of hydrocortisone on the synthesis and secretion of lipids and lipoproteins. Human jejunal explants were cultured for 5 days at 37°C in serum-free medium containing either [¹⁴C]-oleic acid or [¹⁴C]-acetate, alone or supplemented with hydrocortisone (25 or 50 ng/ml). The uptake of [¹⁴C]-oleic acid was associated with the production of triglycerides, phospholipids, and cholesteryl esters, which were all affected by hydrocortisone. This hormonal agent (50 μg) led to the marked reduction of secreted triglycerides (43%, P < 0.01), phospholipids (39%, P < 0.01), and cholesteryl esters (36%, P < 0.05) without altering the characteristic distribution of tissue and medium lipid classes. Similarly, hydrocortisone significantly (P < 0.01) decreased (∼60%) the incorporation of [¹⁴C]-acetate into secreted free and esterified cholesterol in the medium. With [¹⁴C]-oleic acid as a precursor, hydrocortisone significantly diminished the delivery of chylomicrons and very low density lipoproteins to the medium while consistently enhancing the secretion of high density lipoproteins. In parallel, [³⁵S]-methionine pulse-labeling of jejunal explants revealed the concomitant inhibitory effect of hydrocortisone on apo B-100 synthesis and hydrocortisone's stimulatory effect on apo B-48 and apo A-I. These studies suggest that glucocorticoids play a critical role in lipoprotein processing during intestinal development. J. Cell. Biochem. 66:65–76 1997. © 1997 Wiley-Liss, Inc.     

Enhanced Erythrocyte Adhesiveness/Aggregation in Obesity Corresponds to Low‐Grade Inflammation

Objective: Previous studies have suggested that obesity enhances the inflammatory response, producing macromolecules involved in the induction and/or maintenance of increased erythrocyte aggregation. The objectives of this study were to evaluate the correlation between inflammation markers, erythrocyte adhesiveness/aggregation, and the degree of obesity and to assess phosphatidylserine expression on erythrocyte surface membrane of obese vs. nonobese individuals. Research Methods and Procedures: Erythrocyte adhesiveness/aggregation in the peripheral venous blood was evaluated by using a new biomarker, phosphatidylserine expression was assessed by means of flow cytometry, and markers of inflammation were measured in 65 subjects: 30 obese [body mass index (BMI) = 41 ± 7.7 kg/m²] and 35 nonobese (BMI = 24 ± 2.7 kg/m²) individuals. Pearson correlations and Student's t test were performed. Results: A highly significant difference was noted in the degree of erythrocyte adhesiveness/aggregation and markers of inflammation between the study groups. BMI correlated with erythrocyte adhesiveness/aggregation (r = 0.42, p = 0.001), erythrocyte sedimentation rate (r = 0.42, p = 0.001), high‐sensitive C‐reactive protein (r = 0.55, p < 10^?4), fibrinogen (r = 0.37, p = 0.004), and white blood cell count (r = 0.45, p < 10^?4). The degree of erythrocyte adhesiveness/aggregation correlated with erythrocyte sedimentation rate (r = 0.5, p < 10^?4), high‐sensitive C‐reactive protein (r = 0.56, p < 10^?4), fibrinogen (r = 0.54, p < 10^?4), and white blood cell count (r = 0.32, p = 0.01). Discussion: Our results suggest that obesity‐related erythrocyte adhesiveness/aggregation is probably mediated through increased concentrations of adhesive macromolecules in the circulation and not necessarily through hyperlipidemia or phosphatidylserine exposure on erythrocyte's membrane.     

HIV‐1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV‐1)‐infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro‐mitochondrial or anti‐apoptotic strategies as in vitro cell platforms to deal with HIV‐infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt‐PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial‐nuclear encoded subunits II–IV of cytochrome‐c‐oxidase (COXII‐COXIV), respectively, as well as mitochondrial apoptotic events [voltage‐dependent‐anion‐channel‐1(VDAC‐1)‐content and caspase‐9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1‐assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC‐1 and caspase‐9 content were sharply decreased in both chronic HIV‐1‐infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV‐chronicity, to test novel therapeutic options or to study HIV reservoirs.     

CMV seropositivity and T‐cell senescence predict increased cardiovascular mortality in octogenarians: results from the Newcastle 85+ study

Although chronic infection with cytomegalovirus (CMV) is known to drive T lymphocytes toward a senescent phenotype, it remains controversial whether and how CMV can cause coronary heart disease (CHD). To explore whether CMV seropositivity or T‐cell populations associated with immunosenescence were informative for adverse cardiovascular outcome in the very old, we prospectively analyzed peripheral blood samples from 751 octogenarians (38% males) from the Newcastle 85+ study for their power to predict survival during a 65‐month follow‐up (47.3% survival rate). CMV‐seropositive participants showed a higher prevalence of CHD (37.7% vs. 26.7%, P = 0.030) compared to CMV‐seronegative participants together with lower CD4/CD8 ratio (1.7 vs. 4.1, P < 0.0001) and higher frequencies of senescence‐like CD4 memory cells (41.1% vs. 4.5%, P < 0.001) and senescence‐like CD8 memory cells (TEMRA, 28.1% vs. 6.7%, P < 0.001). CMV seropositivity was also associated with increased six‐year cardiovascular mortality (HR 1.75 [1.09–2.82], P = 0.021) or death from myocardial infarction and stroke (HR 1.89 [107–3.36], P = 0.029). Gender‐adjusted multivariate Cox regression analysis revealed that low percentages of senescence‐like CD4 T cells (HR 0.48 [0.32–0.72], P < 0.001) and near‐senescent (CD27 negative) CD8 T cells (HR 0.60 [0.41–0.88], P = 0.029) reduced the risk of cardiovascular death. For senescence‐like CD4, but not near‐senescent CD8 T cells, these associations remained robust after additional adjustment for CMV status, comorbidities, and inflammation markers. We conclude that CMV seropositivity is linked to a higher incidence of CHD in octogenarians and that senescence in both the CD4 and CD8 T‐cell compartments is a predictor of overall cardiovascular mortality as well as death from myocardial infarction and stroke.     

Estado actual de la histoplasmosis diseminada progresiva en pacientes con infección por el VIH en un hospital de tercer nivel en Perú

BackgroundProgressive disseminated histoplasmosis (PDH) is an endemic disease in most of Latin America, especially among patients with HIV. There are few reports about this disease in Peru.AimsTo describe the clinical, epidemiological and mycological features of patients with PDH and HIV evaluated in a tertiary hospital.MethodsA retrospective study to find out the data of patients diagnosed with PDH and HIV in the period 2000–2019 was carried out. For the statistical analysis of quantitative variables, measures of central tendency and dispersion were used; for the qualitative variables, absolute and relative frequencies were used.ResultsForty-three male patients with PDH were diagnosed in the study period, with a median age of 33 years (IQR: 29–38 years) and a median CD₄ lymphocytes count of 39 cells/mm³ (IQR: 20–83 cells/mm³). Eighty six percent of the patients were born or had travelled to the jungle, 58.1% were alcohol users and 16.1% had a history of pulmonary tuberculosis. When compared to histopathology, the culture had a better sensitivity to achieve a diagnosis (p < 0.05).ConclusionsPeruvian patients with PDH and HIV infection were mainly young male adults that were born or had travelled to the jungle, with a CD₄ count below 100 cells/mm³. In patients with the described characteristics it would be advisable to check for PDH. Implementing rapid diagnostic tests is also necessary.